PHACTR1 Antibody

What is PHACTR1 and why is it important in research?

PHACTR1 (Phosphatase and Actin Regulator 1) is a 580 amino acid cytoplasmic protein that plays a crucial role in regulating actin cytoskeleton and is involved in cellular processes such as cell migration and adhesion . It exists in two alternatively spliced isoforms and contains RPEL repeats, indicating its involvement in actin dynamics. The PHACTR1 gene is located on human chromosome 6, a region associated with various diseases including early onset intestinal cancer and porphyria cutanea tarda . PHACTR1's importance in research has escalated following numerous genome-wide association studies that identified single nucleotide polymorphisms (SNPs) at the PHACTR1 locus strongly correlating with coronary artery disease risk . The protein's function in maintaining cellular architecture and facilitating communication between cells makes it essential for tissue integrity and response to external stimuli .

What experimental techniques can detect PHACTR1 protein expression?

The PHACTR1 protein can be detected using multiple experimental techniques, each with specific advantages for different research questions. Western blotting using PHACTR1 antibody provides quantitative data on protein expression levels and can detect the 580 amino acid protein in tissue and cell lysates. Immunoprecipitation allows for isolation of PHACTR1 and associated protein complexes, enabling interaction studies with binding partners such as heat shock protein A8 (HSPA8) . Immunofluorescence microscopy reveals subcellular localization of PHACTR1, which notably shuttles between the nucleus in disturbed flow areas and cytoplasm under laminar flow conditions . Enzyme-linked immunosorbent assay (ELISA) offers sensitive quantification of PHACTR1 in biological samples . For all these methods, the PHACTR1 Antibody (E-2) has demonstrated effectiveness across mouse, rat, and human samples, making it versatile for cross-species studies .

How does PHACTR1 localization change under different flow conditions?

PHACTR1 exhibits distinct localization patterns depending on flow conditions in vascular endothelial cells. Under disturbed flow conditions, PHACTR1 is predominantly localized to the nucleus of endothelial cells, where it can function as a transcriptional corepressor . In contrast, when exposed to laminar flow, PHACTR1 shuttles from the nucleus to the cytoplasm as demonstrated in in vitro studies with human umbilical vein endothelial cells (HUVECs) . This dynamic translocation suggests a mechanism by which PHACTR1 may differentially regulate gene expression depending on hemodynamic conditions. This phenomenon is particularly relevant for atherosclerosis research, as disturbed flow regions in the vasculature are known to be more susceptible to atherosclerotic plaque formation. The nuclear enrichment of PHACTR1 in these regions correlates with its role in promoting endothelial activation and atherosclerosis development . Immunofluorescence microscopy using PHACTR1 antibody is the primary method for visualizing this translocation phenomenon in experimental settings.

What molecular mechanisms link PHACTR1 to coronary artery disease?

PHACTR1 contributes to coronary artery disease through multiple molecular mechanisms centered on endothelial dysfunction. First, PHACTR1 activates the nuclear factor kappa B (NF-κB) pathway, leading to increased expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) . This promotes monocyte adhesion to the endothelium, a critical early step in atherosclerosis. Second, PHACTR1 inhibits Akt/endothelial nitric oxide synthase (eNOS) activation, reducing nitric oxide (NO) production that normally maintains vascular homeostasis . Third, PHACTR1 functions as a peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional corepressor by binding to PPARγ through corepressor motifs . Since PPARγ activation protects against atherosclerosis by inhibiting endothelial activation, PHACTR1's corepressor activity promotes atherosclerosis. Fourth, PHACTR1 interacts with heat shock protein A8 (HSPA8), which has been associated with coronary artery disease and eNOS degradation . Finally, PHACTR1 gene expression is upregulated by pro-inflammatory and pro-atherogenic stimuli, including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and oxidized low-density lipoprotein (oxLDL) .

How does PHACTR1 influence endothelial cell activation?

PHACTR1 promotes endothelial cell activation through several interconnected pathways that culminate in a pro-inflammatory, pro-atherogenic endothelial phenotype. RNA sequencing of endothelial cell-enriched mRNA from PHACTR1 knockout mice revealed that endothelial PHACTR1 depletion affects multiple vascular function pathways . The primary mechanism involves PHACTR1's function as a PPARγ transcriptional corepressor. By binding to PPARγ through corepressor motifs, PHACTR1 inhibits the anti-inflammatory actions of PPARγ in endothelial cells . This repression increases endothelial susceptibility to activation stimuli.

In experimental models, PHACTR1 overexpression disrupts pathways associated with endothelial homeostasis and increases NF-κB-dependent ICAM1 and VCAM1 expression, enhancing monocyte adhesion to the endothelium . This effect can be quantitatively assessed using a monocyte adhesion assay, where THP1 monocytic cells are allowed to adhere to HUVEC monolayers treated with adenovirus expressing PHACTR1 .

Importantly, PHACTR1 also reduces nitric oxide generation by inhibiting Akt/eNOS activation . This dual action of increasing adhesion molecule expression while decreasing protective nitric oxide production creates an environment highly conducive to endothelial dysfunction and atherosclerosis development. Knockout studies confirm these mechanisms, as PPARγ antagonist GW9662 abolishes the protective effects of PHACTR1 deficiency on endothelial cell activation and atherosclerosis in vivo .

What is the role of PHACTR1 in PP1 (Protein Phosphatase 1) complex function?

PHACTR1 forms a complex with Protein Phosphatase 1 (PP1) that significantly alters the phosphatase's substrate specificity and function. The molecular basis for this interaction has been elucidated through crystallographic studies of the Phactr1/PP1 complex . PHACTR1 binding to PP1 occurs through multiple interaction points, with the C-terminal sequences of PHACTR1 docking across PP1's hydrophobic groove, profoundly transforming the surface of PP1 adjacent to its catalytic site .

Key structural features of this interaction include:

• Formation of a deep hydrophobic pocket comprising PHACTR1 residues W542, L545, K550, I553, R554, L557, and H578, and PP1 residues I130, I133, and Y134

• Creation of a narrow amphipathic cavity formed by the positioning of PHACTR1 K561 and R576 across the PP1 hydrophobic groove

• Development of a basic rim from PHACTR1 residues K550, R554, R576, H578, and R579, which radically alters the surface charge distribution

Mutagenesis experiments confirmed the importance of these interactions, with a triple alanine substitution of PHACTR1 L557, F560, and L574 (LFL-3A) resulting in a ~900-fold drop in binding affinity . The acidic substitution L574D reduced binding affinity by 17-fold, while mutations F577A and H578A reduced binding activity by 16-fold and 7-fold, respectively .

This structural remodeling of PP1 by PHACTR1 binding suggests that PHACTR1 can make phosphatases more sequence-specific, potentially directing PP1 activity toward specific substrates relevant to endothelial function and atherosclerosis development .

How can PHACTR1 antibodies be optimized for Western blotting protocols?

For optimal Western blotting results with PHACTR1 antibodies, researchers should implement a comprehensive protocol that addresses several critical factors. Sample preparation is crucial - PHACTR1 is a 580 amino acid cytoplasmic protein , so complete cell lysis using RIPA buffer supplemented with protease inhibitors is recommended to ensure full extraction. When separating proteins, use 8-10% SDS-PAGE gels to achieve optimal resolution around the 65-70 kDa range where PHACTR1 migrates.

During transfer, employ a wet transfer system with 20% methanol for proteins of this size, transferring at 100V for 60-90 minutes or at 30V overnight at 4°C for more complete transfer. For detection, the PHACTR1 Antibody (E-2) can be used at a 1:500-1:1000 dilution, and incubation should occur overnight at 4°C to maximize specific binding . A critical optimization step is blocking - use 5% non-fat dry milk in TBST for 1 hour at room temperature to minimize background.

For difficult samples or when detecting low PHACTR1 expression, consider using PHACTR1 Antibody (E-2) HRP conjugate to eliminate secondary antibody incubation steps and reduce background . Verification of results should include positive controls (cell lines known to express PHACTR1) and negative controls (PHACTR1 knockdown samples). This methodological approach ensures specific and sensitive detection of PHACTR1 in various experimental contexts.

What are the key considerations for PHACTR1 knockdown experiments?

PHACTR1 knockdown experiments require careful design and execution to ensure effective protein depletion while maintaining cell viability and specificity. Based on published protocols, siRNA transfection has been successfully used at a concentration of 20 nM in human umbilical vein endothelial cells (HUVECs) . When implementing this approach, consider the following methodology:

• Transfection optimization: Culture HUVECs in complete medium until 70-80% confluent before transfection. Use a lipid-based transfection reagent and 20 nM PHACTR1 siRNA. Add fresh complete culture medium 4 hours after transfection to minimize cytotoxicity .

• Duration of knockdown: Allow 48 hours post-transfection before collecting RNA or protein for analysis, as this timeframe provides optimal depletion while minimizing off-target effects .

• Controls and validation: Include scrambled siRNA as a negative control and validate knockdown efficiency using Western blotting with PHACTR1 antibody and RT-qPCR for mRNA levels.

• Functional assays: After confirming successful knockdown, assess functional consequences through appropriate assays such as:

  • NF-κB activity using luciferase reporter assays

  • Monocyte adhesion assays to measure endothelial activation

  • Nitric oxide production measurement

  • PPARγ target gene expression analysis

• Phenotype rescue: To confirm specificity, perform rescue experiments by re-expressing siRNA-resistant PHACTR1 constructs.

For in vivo studies, researchers have successfully used global (PHACTR1 knockout) and endothelial cell-specific (PHACTR1 ECKO) knockout mouse models . These models have revealed distinct phenotypes, particularly in atherosclerosis susceptibility, with PHACTR1 deficiency significantly attenuating atherosclerosis in mouse models .

How should researchers approach PHACTR1-PP1 interaction studies?

Investigating PHACTR1-PP1 interactions requires specialized techniques that can detect protein-protein binding and assess its functional consequences. Bio-layer interferometry (BLI) has proven effective for quantifying these interactions, allowing researchers to determine binding affinities and kinetic parameters . A methodological approach using the Octet Red 96 system involves immobilizing His-tagged PP1α (50 μg/ml) on Nickel-coated biosensors followed by incubation with 0.1-10 μM PHACTR1 peptides in appropriate buffer conditions (50 mM Tris pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 0.1% Tween 20, 500 mg BSA/100 ml) .

For mutational analysis studies, researchers should target key residues identified in crystallographic studies. The triple alanine substitution of PHACTR1 L557, F560, and L574 (LFL-3A) produced a dramatic ~900-fold reduction in binding affinity, while the acidic substitution L574D reduced binding by 17-fold . Mutations F577A and H578A reduced binding activity by 16-fold and 7-fold, respectively, providing useful controls for specificity .

Co-immunoprecipitation experiments using PHACTR1 antibody can validate interactions in cellular contexts, while proximity ligation assays can visualize PHACTR1-PP1 interactions in situ. For functional studies, researchers should assess how PHACTR1 binding affects PP1 phosphatase activity using phosphatase assays with various substrates to determine if PHACTR1 alters PP1 substrate specificity as suggested by structural studies .

Additionally, crystallographic studies have provided valuable insights into the molecular details of this interaction. The Phactr1(507-580)/PP1α(7-300) complex has been successfully crystallized using sitting-drop vapor diffusion at 20°C with a well solution of 7.5% PEG 3350 and 0.2 M MgSO4 .

How does PHACTR1 contribute to atherosclerosis development in animal models?

PHACTR1's role in atherosclerosis has been extensively studied using mouse models, revealing unexpected and significant effects on disease progression. Contrary to initial expectations based on genome-wide association studies linking PHACTR1 variants to increased coronary artery disease risk, global PHACTR1 deficiency (PHACTR1 knockout) in apolipoprotein E-deficient (Apoe-/-) mice significantly attenuated atherosclerosis . This protective effect was cell-type specific, with endothelial PHACTR1 promoting atherosclerosis while macrophage PHACTR1 may have different effects.

In methodological terms, atherosclerosis was induced in these models using either a high-fat/high-cholesterol diet for 12 weeks or by partially ligating carotid arteries combined with a 2-week high-fat/high-cholesterol diet . Quantitative assessment of atherosclerosis revealed that:

• Masson trichrome staining showed atherosclerotic plaque areas in the aortic sinus decreased by 22.3% in PHACTR1-/- Apoe-/- mice compared with PHACTR1+/+ Apoe-/- mice .

• En face oil red O staining demonstrated that atherosclerotic lesions decreased by 27.00% in the aortic arch of PHACTR1-/- Apoe-/- mice compared with PHACTR1+/+ Apoe-/- mice, though levels were comparable in the thoracic/abdominal aorta .

• The protective effect was most pronounced in regions of disturbed flow, matching PHACTR1's differential nuclear localization in these areas .

To specifically investigate endothelial PHACTR1's role, researchers generated endothelial cell-specific PHACTR1 knockout (PHACTR1 ECKO) mice crossed with Apoe-/- mice, which also showed significant protection against atherosclerosis development . These findings establish endothelial PHACTR1 as a critical promoter of atherosclerosis, particularly in disturbed flow regions of the vasculature.

What mechanisms underlie PHACTR1's effects on endothelial function in vivo?

PHACTR1 exerts its effects on endothelial function through multiple interconnected molecular mechanisms that collectively promote endothelial dysfunction and atherosclerosis. RNA sequencing of endothelial cell-enriched mRNA from global or EC-specific PHACTR1 knockout mice revealed that PHACTR1 deficiency affected vascular function pathways, with PPARγ identified as the top transcription factor regulating differentially expressed genes .

The primary mechanism involves PHACTR1's function as a PPARγ transcriptional corepressor. PHACTR1 binds to PPARγ through corepressor motifs, inhibiting PPARγ's protective effects against endothelial activation and atherosclerosis . This mechanism was confirmed through multiple experimental approaches:

• PHACTR1 deficiency reduced endothelial activation induced by disturbed flow both in vivo and in vitro .

• Treatment with the PPARγ antagonist GW9662 abolished the protective effects of PHACTR1 knockout on endothelial cell activation and atherosclerosis in vivo, confirming the PPARγ-dependent mechanism .

• PPARγ activation is known to protect against atherosclerosis by inhibiting endothelial activation, and PHACTR1's corepressor activity counteracts this protection .

Additionally, PHACTR1 mediates endothelial inflammation through activating NF-κB-dependent gene expression, particularly increasing ICAM1 and VCAM1 levels that promote monocyte adhesion to the endothelium . PHACTR1 also reduces nitric oxide production by inhibiting Akt/eNOS activation, further compromising endothelial function .

In areas of disturbed flow, PHACTR1 is enriched in the nuclei of endothelial cells, where it can more effectively repress PPARγ transcriptional activity . In contrast, under laminar flow conditions, PHACTR1 shuttles to the cytoplasm, potentially reducing its repressive effects on PPARγ .

What potential therapeutic strategies target PHACTR1 for cardiovascular disease treatment?

Based on the established role of PHACTR1 in promoting endothelial dysfunction and atherosclerosis, several therapeutic strategies targeting this protein show promise for cardiovascular disease treatment. Drug screening studies have identified that several vasoprotective drugs, particularly lipid-lowering statins, decrease PHACTR1 gene expression . This finding suggests that part of statins' beneficial effects may occur through PHACTR1 downregulation, though notably, another lipid-lowering drug, fenofibrate, did not affect PHACTR1 expression .

Given PHACTR1's function as a PPARγ corepressor, PPARγ agonists could theoretically overcome PHACTR1's repressive effects and restore endothelial homeostasis. Since the PPARγ antagonist GW9662 abolished the protective effects of PHACTR1 deficiency in vivo , PPARγ activators may provide targeted benefits in patients with risk-associated PHACTR1 variants.

RNA interference approaches specifically targeting endothelial PHACTR1 represent another potential strategy. The successful use of siRNA (20 nM) to knock down PHACTR1 in human umbilical vein endothelial cells demonstrates feasibility , though delivery challenges would need to be addressed for clinical application.

Small molecule inhibitors designed to disrupt PHACTR1-PPARγ interaction could specifically block PHACTR1's corepressor function without affecting its other cellular roles. The crystal structure of PHACTR1 bound to PP1 reveals a distinctive binding interface , and similar structural studies of PHACTR1-PPARγ interactions could guide inhibitor design.

Finally, targeting PHACTR1's interaction with heat shock protein A8 (HSPA8) represents another avenue, as this interaction has been validated and is associated with coronary artery disease and endothelial nitric oxide synthase degradation . Disrupting this interaction might preserve endothelial nitric oxide production and function.

How do different PHACTR1 isoforms affect experimental outcomes?

PHACTR1 exists in two alternatively spliced isoforms , which can significantly impact experimental outcomes if not properly accounted for in research design. These isoform differences may explain some contradictory findings in the literature regarding PHACTR1's function. When designing experiments, researchers should consider several methodological approaches to address isoform-specific effects:

• Isoform identification: Before conducting functional studies, researchers should determine which PHACTR1 isoforms are expressed in their experimental system using RT-PCR with isoform-specific primers. Western blotting with PHACTR1 antibody may reveal multiple bands corresponding to different isoforms.

• Antibody selection: Verify whether the PHACTR1 antibody used recognizes all isoforms or is isoform-specific. The PHACTR1 Antibody (E-2) detects PHACTR1 protein across species , but its specificity for different isoforms should be confirmed.

• Isoform-specific knockdown: Design siRNA or shRNA constructs that target either common regions (to deplete all isoforms) or isoform-specific sequences. This approach can help dissect isoform-specific functions.

• Selective overexpression: When overexpressing PHACTR1, explicitly state which isoform is being used. For comprehensive studies, compare the effects of overexpressing each isoform separately.

• Context-dependent expression: Different tissues or cellular conditions may favor expression of specific PHACTR1 isoforms. For instance, disturbed flow versus laminar flow conditions might induce different isoform expression patterns in endothelial cells .

Understanding isoform-specific functions is particularly relevant for the development of therapeutic strategies targeting PHACTR1, as targeting one isoform while sparing others might provide more precise intervention with fewer side effects.

What are the emerging techniques for studying PHACTR1's role across different vascular beds?

Advanced technologies are expanding our understanding of PHACTR1's role across diverse vascular beds, which is especially important given that PHACTR1's effects are most pronounced in regions of disturbed flow . Emerging methodological approaches include:

• Single-cell RNA sequencing: This technique can reveal cell-type specific expression patterns of PHACTR1 across different vascular beds. By isolating endothelial cells from various vascular locations (aortic arch, thoracic aorta, resistance vessels) in wild-type and PHACTR1 knockout mice, researchers can map PHACTR1's differential expression and correlate it with region-specific gene signatures.

• Intravital microscopy: This approach allows real-time visualization of endothelial-leukocyte interactions in living animals. By combining this with endothelial-specific PHACTR1 knockout models, researchers can directly observe how PHACTR1 affects leukocyte adhesion and transmigration in different vascular beds under physiological flow conditions.

• Organ-on-chip technologies: Microfluidic devices that recapitulate vascular microenvironments with controlled flow patterns can help dissect how PHACTR1 responds to different hemodynamic conditions. These systems can model diverse vascular beds and apply specific flow patterns (laminar, disturbed, oscillatory) while monitoring PHACTR1 localization and function.

• CRISPR-Cas9 genomic editing: Beyond conventional knockout approaches, CRISPR-Cas9 can introduce specific SNPs associated with coronary artery disease into the PHACTR1 locus, allowing researchers to study how these variants affect PHACTR1 function in different vascular beds.

• Optical coherence tomography and other in vivo imaging: These non-invasive techniques can assess vascular structure and function in PHACTR1 knockout models across multiple vascular beds simultaneously, providing whole-organism perspectives on PHACTR1's role in vascular homeostasis.

These emerging approaches will help clarify why PHACTR1's effects on atherosclerosis vary between the aortic arch (27% reduction in lesion area in knockout mice) and the thoracic/abdominal aorta (comparable lesion area) , potentially informing more targeted therapeutic strategies.

What contradictions exist in the PHACTR1 literature and how might they be resolved?

The PHACTR1 literature contains several notable contradictions that require resolution through methodological refinement and integration of diverse experimental approaches. One central contradiction involves PHACTR1's role in atherosclerosis. While genome-wide association studies consistently link PHACTR1 variants to increased coronary artery disease risk, experimental models show that PHACTR1 deficiency attenuates atherosclerosis in mouse models . This apparent paradox might be explained through several approaches:

Integration of in vitro, in vivo, and human data through systems biology approaches could help resolve these contradictions. Meta-analyses combining results from diverse experimental systems while accounting for methodological differences would provide a more comprehensive understanding of PHACTR1's complex role in cardiovascular health and disease.